Looking at both the epidemiological and pharmacological research enterolactone stands out as key to the activity in vivo for lignans. In fact looking at pharmacological research it seems unlikely that circulating plasma levels of the parent lignans reach EC50 values (active concentrations) in humans. On the other hand enterolactone at the concentrations achieved in our human clinicals with HMR are well with the active EC50 values. Enterolactone have four pharmacological properties in that it is weakly estrogenic, a potent anti-inflammatory, strongly antioxidant and an enzymatic inhibitor.
Looking at the epidemiological studies it is plasma enterolactone not parent lignans that are associated with the health benefits. I have included references as below.
Hope this assist.
Robin Ward
Epidemiological references for heart health.
1. Arch Intern Med. 2003 May 12;163(9):1099-104. Risk of cardiovascular disease-related and all-cause death according to serum concentrations of enterolactone: Kuopio Ischaemic Heart Disease Risk Factor Study. Vanharanta M, Voutilainen S, Rissanen TH, Adlercreutz H, Salonen JT. Research Institute of Public Health, University of Kuopio, Harjulantie 1B, 70210 Kuopio, Finland.
2. J Nutr. 2002 Feb;132(2):276-82. Dietary intake of phytoestrogens is associated with a favorable metabolic cardiovascular risk profile in postmenopausal U.S.women: the Framingham study. de Kleijn MJ, van der Schouw YT, Wilson PW, Grobbee DE, Jacques PF. Julius Center for General Practice and Patient Oriented Research, University Medical Center, Utrecht, The Netherlands.
3. Atherosclerosis. 2002 Feb;160(2):465-9. Association between low serum enterolactone and increased plasma F2-isoprostanes, a measure of lipid peroxidation. Vanharanta M, Voutilainen S, Nurmi T, Kaikkonen J, Roberts LJ, Morrow JD, Adlercreutz H, Salonen JT. Research Institute of Public Health, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland.
4. Lancet. 1999 Dec 18-25;354(9196):2112-5. Risk of acute coronary events according to serum concentrations of enterolactone: a prospective population-based case-control study. Vanharanta M, Voutilainen S, Lakka TA, van der Lee M, Adlercreutz H, Salonen JT. Research Institute of Public Health, University of Kuopio, Finland.
5. Arterioscler Thromb Vasc Biol. 2002 Aug 1;22(8):1316-22. Higher usual dietary intake of phytoestrogens is associated with lower aortic stiffness in postmenopausal women. van der Schouw YT, Pijpe A, Lebrun CE, Bots ML, Peeters PH, van Staveren WA, Lamberts SW, Grobbee DE. Julius Center for Health Sciences and Primary Care, University Medical Center, Utrecht, The Netherlands.
Epidemiological references for breast health.
1. Cancer Epidemiol Biomarkers Prev. 2001 Apr;10(4):339-44. Serum enterolactone and risk of breast cancer: a case-control study in eastern Finland. Pietinen P, Stumpf K, Mannisto S, Kataja V, Uusitupa M, Adlercreutz H. Department of Nutrition, National Public Health Institute, Mannerheimintie 166, 00300 Helsinki, Finland.
pirjo.pietinen@ktl.fi
2. Lancet. 1997 Oct 4;350(9083):990-4. Case-control study of phyto-oestrogens and breast cancer. Ingram D, Sanders K, Kolybaba M, Lopez D. University Department of Surgery, Queen Elizabeth II Medical Centre, Perth, Western Australia.
3. Cancer Causes Control. 2004 Oct;15(8):805-18. Phyto-oestrogen intake and breast cancer risk in South Asian women in England: findings from a population-based case-control study. dos Santos Silva I, Mangtani P, McCormack V, Bhakta D, McMichael AJ, Sevak L. Department of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
isabel.silva@lshtm.ac.uk.
4. Cancer Epidemiol Biomarkers Prev. 2004 Dec;13(12):2084-9. Plasma enterolactone and breast cancer incidence by estrogen receptor status. Olsen A, Knudsen KE, Thomsen BL, Loft S, Stripp C, Overvad K, Moller S, Tjonneland A. Institute of Cancer Epidemiology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen O, Denmark.
anja@cancer.dk.
5. Int J Cancer. 2004 Sep 1;111(3):440-3. Dietary lignan intakes and risk of pre- and postmenopausal breast cancer. McCann SE, Muti P, Vito D, Edge SB, Trevisan M, Freudenheim JL. Department of Epidemiology, Division of Cancer Prevention and Population Sciences, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
susan.mccann@roswellpark.org.
Proprietary/Specific pharmacological data on HMR/Enterolactone.
The majority of the studies were commissioned/conducted by Linnea/Hormos/Turku University (Finland)/Abo University (Finland).
Published.
Eur J Cancer Prev. 2002 Aug;11 Suppl 2:S48-57. Antioxidant and antitumor effects of hydroxymatairesinol (HM-3000, HMR), a lignan isolated from the knots of spruce.
Kangas L, Saarinen N, Mutanen M, Ahotupa M, Hirsinummi R, Unkila M, Perala M, Soininen P, Laatikainen R, Korte H, Santti R.
Hormos Nutraceutical Ltd, Turku, Finland.
The antioxidant properties of hydroxymatairesinol (HM-3000) were studied in vitro in lipid peroxidation, superoxide and peroxyl radical scavenging, and LDL-oxidation models in comparison with the known synthetic antioxidants Trolox (a water-soluble vitamin E derivative), butylated hydroxyanisol (BHA) and butylated hydroxytoluene (BHT). On a molar basis HM-3000 was a more effective antioxidant than Trolox in all assays and more effective than BHT or BHA in lipid peroxidation and superoxide scavenging test. The in vivo antioxidative effect (evaluated as the weight gain of C57BL/6J mice fed an alpha-tocopherol-deficient diet) of HM-3000 (500 mg/kg per day) was comparable to that of DL-alpha-tocopherol (766 mg/kg per day). The antitumor activity of HM-3000 was studied in dimethylbenz[a]anthracene (DMBA)-induced rat mammary cancer. HM-3000 had a statistically significant inhibitory effect on tumor growth. Prevention of tumor formation was also evaluated in the Apc(Min) mice model, which develops intestinal polyps spontaneously. HM-3000 was given in diet at 30 mg/kg per day and decreased the formation of polyps and prevented beta-catenin accumulation into the nucleus, the pathophysiological hallmark of polyp formation in this mouse model. In short-term toxicity studies (up to 28 days) HM-3000 was essentially non-toxic when given p.o. to rats and dogs (daily doses up to 2000 and 665 mg/kg, respectively); HM-3000 was shown to be well absorbed (> 50% of the dose) and rapidly eliminated. In human studies HM-3000 has been given in single doses up to 1350 mg to healthy male volunteers without treatment-related adverse events. Rapid absorption from the gastrointestinal tract and partial metabolism to enterolactone in humans was demonstrated. In summary, HM-3000 is a safe, novel enterolactone precursor lignan with antioxidant and antitumor properties.
Mol Cancer Ther. 2002 Aug;1(10):869-76. Enterolactone inhibits the growth of 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in the rat.
Saarinen NM, Huovinen R, Warri A, Makela SI, Valentin-Blasini L, Sjoholm R, Ammala J, Lehtila R, Eckerman C, Collan YU, Santti RS.
Institute of Biomedicine, Department of Anatomy, University of Turku, FIN-20520 Turku, Finland.
The inverse association between a high enterolactone (ENL) concentration in both urine and serum, and the risk of breast cancer found in epidemiological studies suggests a chemopreventive action for ENL. However, no causal relationship has been established in clinical studies or in experimental models for breast cancer. In the present study, the potential chemopreventive action of p.o. administered ENL (1 or 10 mg/kg of body weight) was tested in 7,12-dimethylbenz(a)anthracene-induced mammary cancers of the rat. Rats were maintained on a standard open-formula chow diet. Daily p.o. administration of ENL at a dose of 10 mg/kg of body weight for 7 weeks significantly inhibited tumor growth. The growth-inhibitory effect of ENL was more pronounced on the new tumors, which developed during the treatment period, but ENL also inhibited the growth of those tumors established before the start of the lignan administration. The rat serum concentration of ENL, which illustrated a permanent positive effect on breast cancer growth, was 0.4 microM, which is >10-fold as compared with the serum concentrations found in the general human population. The effect of ENL was not restricted to any specific histological tumor type. ENL was demonstrated to act as a weak aromatase inhibitor in vitro and to reduce the relative uterine weight of the 7,12-dimethylbenz(a)anthracene-treated nonovariectomized rats. However, in a short-term assay ENL had no effect on the uterine growth of the intact or androstenedione-treated immature rats. Thus, the mechanism of the ENL action and its minimum or optimal daily dose remains to be clarified.
Exp Biol Med (Maywood). 2005 Mar;230(3):217-23. Anticancer effects of a plant lignan 7-hydroxymatairesinol on a prostate cancer model in vivo.
Bylund A, Saarinen N, Zhang JX, Bergh A, Widmark A, Johansson A, Lundin E, Adlercreutz H, Hallmans G, Stattin P, Makela S.
University of Turku, Functional Foods Forum, FI-20014 Turku, Finland.
Clinical intervention studies and experimental studies with lignan-rich diets suggest that lignans may have inhibitory effects on prostate cancer, but no clinical or experimental studies with purified lignans have been published. The purpose of this study was to investigate the effect of a plant lignan 7-hydroxymatairesinol (HMR) on LNCaP human prostate cancer xenografts in athymic mice. Athymic nude male mice were injected subcutaneously with LNCaP cells. Starting 3 days after tumor cell injections, a control diet or a control diet supplemented with 0.15% or 0.30% of HMR was administered to mice and the tumor take rate and growth was observed for 9 weeks. HMR diet inhibited the growth of LNCaP tumors. Mice treated with HMR had smaller tumor volume, lower tumor take rate, increased proportion of nongrowing tumors, and higher tumor cell apoptotic index compared with controls. Furthermore, the cell proliferation index was reduced in mice receiving the 0.30% HMR diet compared with mice receiving the control diet. Our results suggest that dietary HMR started at the early phase of the tumor development inhibits the growth of the LNCaP human prostate cancer xenografts in athymic male mice.
Regul Toxicol Pharmacol. 2005 Feb;41(1):28-38. Epub 2004 Dec 9. A thirteen week dietary toxicity study with 7-hydroxymatairesinol potassium acetate (HMRlignan) in rats.
Lina B, Korte H, Nyman L, Unkila M.
TNO Nutrition and Food Research, Department of Toxicology and Applied Pharmacology, Zeist, The Netherlands.
Plant lignan 7-hydroxymatairesinol (7-HMR) is a novel precursor of the mammalian lignan enterolactone. A 13 week toxicity study at dietary levels of 0, 0.25, 1, and 4% (w/w) of potassium acetate complex of 7-HMR (HMRlignan) was conducted in the Wistar rat. These dietary levels resulted in an average daily intake of 160, 640, and 2600 mg HMRlignan/kg body weight/day, respectively. A considerable systemic exposure of HMRlignan was verified by dose-related increases in plasma total (conjugated and unconjugated) concentration of 7-HMR and metabolites enterolactone, 7-hydroxyenterolactone, and matairesinol. Enterolactone appeared to be the major metabolite. Most (>96%) of the circulating 7-HMR and enterolactone was in conjugated form as measured from the low-dose rat plasma samples. HMRlignan exposure did not significantly affect clinical signs, ophthalmoscopy or neurobehavioural observations, and motor activity. Transient reductions in food intake and body weight gain in the mid-and high-dose group were ascribed to decreased palatability of the test feed. Only in males of the high-dose group the body weights remained slightly reduced throughout the study. In the high-dose group the number of thrombocytes (females), and total white blood cell count (males) were increased. Plasma triglycerides were dose-dependently depressed in males of all test groups and in females of the mid- and high-dose group, while plasma total cholesterol, and phospholipids were decreased in high-dose males. These changes, which have also been reported for other (flaxseed) lignans, were not considered to represent adverse effects. The relative weight of the kidneys was increased in males of the high-dose group. The weight of the full and empty caecum showed dose-related increases in males of all treatment groups and in females of the high-dose group. Absolute ovary weights were decreased in all treatment groups while decreases in relative ovary weights were confined to the mid- and high-dose group. In addition, a marginal lengthening of the estrus cycle was noted in high-dose females. Apart from prevention of hyaline droplet nephropathy in all high-dose male rats, there were no treatment-related histopathological alterations. It was concluded that HMRlignan showed weak antiestrogen-like activity which may be mediated through enterolactone metabolite. Based on declined ovary weight, the no observed adverse effect level of HMRlignan was set at 0.25% in feed corresponding to 160 mg/kg body weight/day.
Org Biomol Chem. 2005 Sep 21;3(18):3336-47. Epub 2005 Aug 9. Chemical studies on antioxidant mechanisms and free radical scavenging properties of lignans.
Eklund PC, Langvik OK, Warna JP, Salmi TO, Willfor SM, Sjoholm RE.
Department of Organic Chemistry, Process Chemistry Centre, Abo Akademi University, Biskopsgatan 8, FIN-20500, Abo, Finland.
paeklund@abo.fi rsjoholm@abo.fi
The antioxidant activity, in terms of radical scavenging capacity, of altogether 15 different lignans was measured by monitoring the scavenging of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). The effect of differences in skeletal arrangement or the degree of oxidation of the lignans was investigated in a structure-activity relationship study. A large variety in the radical scavenging capacities of the different lignans was observed and related to some structural features. Lignans with catechol (3,4-dihydroxyphenyl) moieties exhibited the highest radical scavenging capacity, while the corresponding guaiacyl (3-methoxy-4-hydroxyphenyl) lignans showed a slightly weaker scavenging capacity. In addition, the butanediol structure was found to enhance the activity, whereas a higher degree of oxidation at the benzylic positions decreased the activity. Additionally, the readily available lignans (-)-secoisolariciresinol, a mixture of hydroxymatairesinol epimers and (-)-matairesinol were studied in more detail, including kinetic measurements and identification of oxidation products in the reactions with DPPH and ABAP (2,2-azobis(2-methylpropionamidine) dihydrochloride. The identification of reaction products, by GC-MS, HPLC-MS and NMR spectroscopy, showed that dimerisation of the two aromatic moieties was the major radical termination reaction. Also, the formation of adducts was a predominant reaction in the experiments with ABAP. The kinetic data obtained from the reactions between the lignans and DPPH indicated a complex reaction mechanism.
J Steroid Biochem Mol Biol. 2005 Feb;93(2-5):209-19. Epub 2005 Feb 1. Structural determinants of plant lignans for growth of mammary tumors and hormonal responses in vivo.
Saarinen NM, Penttinen PE, Smeds AI, Hurmerinta TT, Makela SI.
Functional Foods Forum, University of Turku, Itainen Pitkakatu 4A, FI-20520 Turku, Finland.
nisaarin@utu.fi
Low risk of breast cancer (BC) has been proposed to be associated with high intake of lignans. Some plant lignans are converted to mammalian lignans, e.g., enterolactone (ENL), suggested to be the biologically active lignan forms. Until now, little attention has been paid to the possible biological activities of plant lignans, even though some plant lignans are absorbed and present in serum and urine. In this study, we have investigated the antitumorigenic and endocrine-modulatory activities of different plant lignans in order to clarify the structure-activity relationships. 7-Hydroxymatairesinol (HMR) is converted to ENL, and both HMR and ENL inhibit the growth of 7,12-dimethylbenz[a]-anthracene (DMBA)-induced mammary cancer. Nortrachelogenin (NTG) resembles HMR, but has a hydroxyl group at C-8 instead of C-7 and is not converted to ENL. In DMBA-model, NTG showed no inhibition of tumor growth, but increased the uterine weight. Furthermore, life-long exposure to NTG increased uterine weight in immature females and ventral prostate weight in adult males. In contrast, life-long exposure to HMR had no effects on uterine or prostate weights at any age. Our results indicate that a difference in the position of one hydroxyl group results in distinct biological responses in vivo, as well as different lignan metabolite profiles.
Exp Biol Med (Maywood). 2004 May;229(5):417-24.
HMR/Enterolactone specific but non-proprietry or not commission by Linnea/Hormos/Turku University (Finland)/Abo University (Finland).
Chemopreventive effects of hydroxymatairesinol on uterine carcinogenesis in Donryu rats.
Katsuda S, Yoshida M, Saarinen N, Smeds A, Nakae D, Santti R, Maekawa A.
Department of Biological Safety Research, Japan Food Research Laboratories, Tama-shi, Tokyo 206-0025, Japan.
katudas@jfrl.or.jp
Hydroxymatairesinol (HMR), obtained from the heartwood of spruce (Picea abies), has been demonstrated to exert chemo-preventive effects on the development of mammary tumors in rats. To examine the influence of HMR on uterine carcinogenesis, adult Donryu rats were initiated with a single intrauterine treatment of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 11 weeks of age and fed thereafter 0, 200, or 600 ppm HMR mixed in the soy-containing diet until 15 months of age. Incidences of uterine adenocarcinoma in both 200 and 600 ppm HMR-dosed groups were significantly reduced to 11% and 15%, respectively, less than 50% of 0 ppm, at the end of the experiment (P < 0.05). A delay in the start of persistent estrus by HMR was observed at 8 months of age compared with controls given carcinogen alone. From urinalysis, HMR was metabolized mainly to enterolactone and hydroxyenterolactone. These findings suggest that HMR or its metabolites exert chemo-preventive effects in the rat ENNG-uterine carcinogenesis model.
Cancer Epidemiol Biomarkers Prev. 2005 Sep;14(9):2154-9. Plasma levels of enterolactone and percentage mammographic density among postmenopausal women.
Stuedal A, Gram IT, Bremnes Y, Adlercreutz H, Veierod MB, Ursin G.
Department of Nutrition, University of Oslo, Norway.
anne.stuedal@medisin.uio.no
AIMS: Certain phytoestrogens, such as lignans, may protect against developing breast cancer. Enterolactone is a lignan metabolite produced by the intestinal flora from dietary precursors such as whole grains, vegetables, and fruits. Enterolactone has been shown to have weak estrogenic and antiestrogenic properties. We decided to examine the association between plasma levels of enterolactone and mammographic density, a biomarker for breast cancer risk. METHODS: We included data from postmenopausal women ages 55 and older who participated in a cross-sectional mammogram study in Tromso, Norway. Mammograms, plasma enterolactone measurements, as well as information on anthropometric and hormonal/reproduction factors were available on 616 women. We assessed mammographic density using a previously validated computer-assisted method. We estimated correlation coefficients and conducted multiple regression analyses. RESULTS: Mean mammographic density increased slightly across quartiles of enterolactone; the women in the highest quartile had, on average, 3.1% (absolute difference) higher percentage mammographic density compared with the lowest quartile (P(trend) < 0.01). After adjustment for age, body mass index, number of full-term pregnancies, age at first birth, and use of postmenopausal hormone therapy, the mean difference in density was reduced to 2.0% (P(trend) = 0.05). Results were similar when restricted to the 454 current hormone nonusers. The fully adjusted statistical model explained 28.3% of the total variability in mammographic percentage density, with body mass index contributing 18.2% and enterolactone only 0.9%. CONCLUSION: In our study, higher levels of enterolactone were associated with slightly higher percentage mammographic density. Our results suggest that if higher enterolactone levels reduce the risk of developing breast cancer in postmenopausal women, then this effect is not through lowering mammographic density.
Anticancer Res. 2005 May-Jun;25(3B):2269-76. Enterolactone induces apoptosis and inhibits growth of Colo 201 human colon cancer cells both in vitro and in vivo.
Danbara N, Yuri T, Tsujita-Kyutoku M, Tsukamoto R, Uehara N, Tsubura A.
Department of Pathology II, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan.
BACKGROUND: The mammalian lignan enterolactone (ENL) is produced from plant lignans which are present in large amounts in flaxseed (linseed). The effect of ENL on colon cancer cell growth in vitro and in vivo, and its mechanisms of action, have not been studied in detail. MATERIALS AND METHODS: The growth of the colo 201 human colon cancer cell line was examined by colorimetric 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2- (4-sulphophenyl)-2H-tetrazolium (MTS) assay, while the expression of apoptosis- and proliferation-related proteins (p53, Bax, Bcl-xL and S, Bcl-2, Caspase-8, Caspase-3 and proliferating cell nuclear antigen (PCNA)) were examined by Western blotting. In vivo tumor growth was examined by transplanting colo 201 cells into ENL-treated and placebo-treated athymic mice. RESULTS: The MTS assay showed that ENL suppressed colo 201 cell growth (IC50 for 72 h: 118.4 microM) in vitro. On flow cytometry, induction of apoptosis was confirmed by the appearance of subG1 populations, while cell cycle progression was not affected. The expression of an apoptosis-suppressing protein (Bcl-2) was down-regulated, an apoptosis-enhancing protein (cleaved form of Caspase-3) was up-regulated, proliferation-related PCNA protein was down-regulated and p53, Bax, Bcl-xL and S and Caspase-8 levels were unchanged. ENL, at a dose of 10 mg/kg given 3 times per week by subcutaneous injection, significantly inhibited the growth of colo 201 cells transplanted into athymic mice without any adverse effects. CONCLUSION: ENL suppressed colo 201 human colon cancer cell growth both in vitro and in vivo. The tumor-suppressing mechanisms included apoptosis and decreased cell proliferation.
Cancer Epidemiol Biomarkers Prev. 2004 Dec;13(12):2084-9. Plasma enterolactone and breast cancer incidence by estrogen receptor status.
Olsen A, Knudsen KE, Thomsen BL, Loft S, Stripp C, Overvad K, Moller S, Tjonneland A.
Institute of Cancer Epidemiology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen O, Denmark.
anja@cancer.dk
The phytoestrogen enterolactone has been hypothesized to prevent breast cancer. Because one of the biological effects of enterolactone is probably estrogenic, it is possible that the preventive effect on breast cancer differs with the estrogen receptor (ER) alpha status of the tumor. The objective of this study was to investigate whether high plasma levels of enterolactone are associated with breast cancer risk and whether the ERalpha status of the tumor influences this relation. The cohort study Diet, Cancer and Health included 29,785 women, ages 50 to 64 years, between 1993 and 1997. Information about diet and life-style factors was obtained by questionnaire, and blood was drawn from each participant. We matched 381 postmenopausal breast cancer cases to 381 controls and analyzed the concentration of enterolactone in plasma with a time-resolved fluoroimmunoassay. Associations between plasma concentrations of enterolactone and breast cancer were analyzed by logistic regression. The incidence rate ratio (IRR) for all breast cancer was 0.93 [95% confidence interval (CI), 0.86-1.01] per 20 nmol/L higher plasma concentration of enterolactone. For ERalpha-positive cancers (n=273) only a weak association was seen (IRR, 0.97; 95% CI, 0.88-1.06), whereas for ERalpha-negative cancers (n = 80; IRR, 0.71; 95% CI, 0.53-0.94) a protective effect was seen per 20 nmol/L higher plasma enterolactone. In accordance with earlier research, we found a tendency toward a lower risk for breast cancer with higher concentrations of enterolactone, which was restricted almost entirely to ERalpha-negative breast cancer.
